GLP-1 derivative and preparation thereof absorbable via mucous membrane

ABSTRACT

A GLP-1 derivative is provided including an amino acid sequence of GLP-1 (7-35) having deletion, substitution and/or addition of one or more amino acids and having Waa-(Xaa)n-Yaa (in which Waa is Arg or Lys, Xaa is Arg or Lys, n is an integer of 0 to 14, and Yaa is Arg, Arg-NH 2 , Lys, Lys-NH 2  or Hse) added to the C-terminus of the peptide having a GLP-1 activity. These derivatives are highly absorbable via a mucous membrane. The GLP-1 derivative can be conferred with resistance to dipeptidyl peptidase IV by substituting amino acid 8 in its GLP-1 amino acid sequence with Ser, or with resistance to trypsin by substituting amino acids 26 and 34 with Gln and Asn, respectively. 
     The absorption efficiency of the GLP-1 derivatives via mucous membranes can be further improved by preparing a composition using a charge-regulated fat emulsion regulated to be negatively charged thereon.

CROSS REFERENCE TO RELATED APPLICATION

This application is a National Phase of International Application No.PCT/JP2003/013020 having an international filing date of Oct. 10, 2003,published in Japanese on May 6, 2004, which claims the benefit ofJapanese Application 2002-299283, filed Oct. 11, 2002, the entirety ofwhich is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to novel human glucagon-like peptide-1(GLP-1) derivatives absorbed highly via a mucous membrane in the oralcavity, lung, nose or intestines, production thereof and a method ofusing the same.

BACKGROUND ART

GLP-1 (Glucagon-Like Peptide-1) is known as an incretin hormone which issecreted from digestive tracts upon ingestion of food to act on thepancreas and stimulate insulin secretion. As a hormone exhibiting asimilar action, there is GIP (Gastric Inhibitory Polypeptide orGlucose-dependent Insulinotropic Polypeptide). This incretin effect issuggested to be absent or reduced inpatients with type 2 diabetes,compared with healthy persons, and this is considered as one of causeshigh blood glucose. For example, it is reported that inpatients withtype 2 diabetes, blood GLP-1 level is lowered, while blood GIP level isnormal. As a result of administering the incretin hormones to patientswith type 2 diabetes, there is no difference upon the insulinsecretion-stimulating activity of GLP-1 between the patients and healthypersons, while the insulin secretion-stimulating activity of GIP issignificantly lower in the patients than healthy persons. Accordingly,the response of the patients with diabetes to GLP-1 is maintained; thus,a GLP-1 preparation compensating for its shortage can be expected toserve as a medicine for treatment of diabetes.

The action of GLP-1 on insulin secretion is characterized by glucoselevel dependent that GLP-1 does not stimulate insulin secretion in theblood glucose level of 110 mg/dL or less. That is, Administration ofGLP-1 has clinical advantages that lower possibility of hypoglycemia,and suppress the excessive insulin secretion so that the exhaustion ofthe pancreas is is prevented. While A sulfonylurea, used mainly intreatment of type 2 diabetes, closes ATP-sensitive K⁺ channelscontinuously to promote insulin secretion it causes low blood glucose,exhaustion of the pancreas by excessive stimulation of β cells, andsecondary failure in administration for a long period of time.Accordingly, the pharmacological characteristics of GLP-1 are veryuseful and different from those of the conventional medicine fordiabetes.

GLP-1 also have the following characteristics: suppression of glucagonsecretion, delay of gastric emptying, suppression of stomach acidsecretin, action on the brain to suppress appetite, promotion of insulinsynthesis in pancreatic β cells and proliferation of pancreatic β cells.Therefore, GLP-1 is considered not only effective for treatment ofdiabetes by antagonizing the cause of high blood glucose such ashyperglucagonemia in type 2 diabetes, but also effective for treatmentof obesity.

However, as GLP-1 is the polypeptide made up of 30 or 31 amino acids, itis digested upon oral ingestion and decomposed by digestive enzyme inthe digestive tract, and is thus not absorbed. The administrationthereof by intravenous injection or subcutaneous injection of GLP-1 isattempted at present. Further, it is known that GLP-1 undergoesdecomposition with dipeptidyl peptidase IV (DPPIV) present in blood ortissues so that the half-life thereof in the living body is as veryshort as 1 to 2 minutes, thus giving rise to an obstacle to clinicalapplications.

To solve these problems, some researches and developments have beenmade. It is attempted to develop, for example, its derivative having anamino acid sequence substituted at position 8 which is hardly degradableto attain a longer half-life (Diabetologia 41: 271-278 (1998) andBiochem 40: 2860-2869 (2001)) and a sustained-release injection showingsustained subcutaneous absorption. An injection of lizard-derived,synthetic Exendin-4 having a GLP-1 like agonistic activity and a longhalf-life in blood (Am J Physiol 281: E155-E161 (2001)) is alsodeveloped. However, an administration route other than via an injectionis desired for wide application of GLP-1 as a medicine for diabetes andin consideration of patients' burden and convenience.

As an administration method not using an invasive means such as aninjection or a preparation for oral administration, use of a preparationfor absorption through mucous membranes in the lung, oral cavity, nasalcavity, vagina, eye, rectum and the like is available. But a peptidesuch as GLP-1 upon being administered alone, is poor in absorption via amucous membrane. Accordingly, a polymer such as peptide is formulatedtogether with an absorption promoter. To secure sustained absorption ofchemicals, the preparation makes use of a water-soluble orwater-swelling binder, and is formulated in the form of film adhering tothe skin layer, buccal tablets, ointment or troche. Many absorptionpromoters or binders have been examined and found to be effective infacilitating administration of chemicals via mucous membranes. However,as the absorption rate, in terms of bioavailability, of GLP-1 in buccaltablets containing 400 μg GLP-1 through a mucous membrane in the humanoral cavity reported by Gutniak et al. (“Diabetes Care” 20: 1874-1879(1997)), even by using the prior art described above, is 7% of theabsorption of its intravenous injection or 47% of the absorption of itssubcutaneous injection, and its absorptivity is not sufficient.

Dipeptidyl peptidase IV is known as an enzyme degradating of GLP-1,which is widely distributed not only in the kidney, liver, smallintestine, salivary gland and various connecting tissues, but also inbody fluid such as blood, urine and saliva and in a mucous membrane inthe nasal cavity, and also possibly in other mucous membrane tissues.

DISCLOSURE OF INVENTION

Absorption of GLP-1 via mucous membrane, as compared with absorption byan injection, is considerably ineffective due to low membranepermeability and degradation in absorption sites. Though the nasaladministration of GLP-1 as such is not impossible a very large dose ofGLP-1 is necessary for achieving a sufficient pharmacological effect.Accordingly, development of naturally occurring GLP-1 as apharmaceutical preparation for nasal administration is unrealistic fromthe viewpoint of the production cost of GLP-1 peptide. For clinicalapplication of GLP-1, it is necessary to develop GLP-1 derivatives whoseabsorptivity via mucous membranes is comparative to that of aninjection. Accordingly, the present inventors anticipated novel GLP-1derivatives with improved absorptivity via mucous membranes, and madeextensive studies to provide a preparation for mucous membraneadministration as a substitute for an injection.

As a result, the inventors arrived at a novel idea that the absorptionof GLP-1 via mucous membranes can be increased by adding positivelycharged arginine or lysine. In addition, they anticipated addition ofseveral residues of arginine and/lysine to the C-terminus thereof whilethe N-terminus important for expressing the activity was kept intact, toobtain GLP-1 derivatives described below. For further increasingabsorptivity via mucous membranes, a charge-regulated fat emulsionhaving a surface regulated to be negatively charged was used to devise aGLP-1 preparation with significant improvement in mucous membraneabsorption.

That is, the GLP-1 derivative of the present invention is a peptidecomprising an amino acid sequence of GLP-1 (7-35) having deletion,substitution and/or addition of one or a few amino acids and having aGLP-1 activity with Waa-(Xaa)n-Yaa (in which Waa is Arg or Lys, Xaa isArg or Lys, n is an integer of 0 to 14, and Yaa is Arg, Arg-NH₂, Lys,Lys-NH₂ or Hse) on its C-terminus of the peptide. As described above, anovel GLP-1 derivative showing high absorption via mucous membranes,that is, high bioavailability via mucous membranes, can be provided byadding several residues of arginine and/or lysine to the C-terminusthereof.

To confer resistance to dipeptidyl peptidase IV, amino acid in position8 of the GLP-1 derivative of the present invention is preferably serine.Such peptide is represented by the general formula: [Ser⁸]-GLP-1(7-35)-Waa-(Xaa)n-Yaa (in which Waa is Arg or Lys, Xaa is Arg or Lys, nis an integer of 0 to 14, and Yaa is Arg, Arg-NH₂, Lys, Lys-NH₂ or Hse).

The GLP-1 derivative of the present invention can be endowed withtrypsin resistance by substituting lysine at position 26 with glutamineand lysine at position 34 with asparagine. Such peptide is representedby the general formula: [Gln²⁶, Asn³⁴]-GLP-1(7-35)-Waa-(Xaa)n-Yaa (inwhich Waa is Arg or Lys, Xaa is Arg or Lys, n is an integer of 0 to 14,and Yaa is Arg, Arg-NH₂, Lys, Lys-NH₂ or Hse).

As a matter of course, these dipeptidyl peptidase IV-resistant ortrypsin-resistant GLP-1 derivatives can also have deletion, substitutionand/or addition of one or more amino acids in the amino acid sequence ofGLP-1 (7-35).

In the GLP-1 derivatives of the present invention, n is preferably aninteger of 1 to 9, more preferably an integer of 3 to 5.

The most preferable peptide among the GLP-1 derivatives of the presentinvention is represented by the general formula: [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1 (7-35)-(Arg)n-Yaa (in which n is an integer of 4 to 6, andYaa is Arg or Arg-NH₂).

The efficiency of absorption of the GLP-1 derivative was examined byadministering the GLP-1 derivative of the present invention into micevia the nose, and then measuring its blood glucose lowering activity andinsulin secretion stimulating activity in the mice in a oral glucosetolerance test. As a result, the GLP-1 derivative showed a high bloodglucose depressing action and insulin secretion promoting action, andthe GLP-1 derivative of the invention in an amount of 1/10 relative tonaturally occurring GLP-1 showed a similar effect to that of thenaturally occurring GLP-1, and it is thus estimated that the absorptionvia the nasal mucous membrane is 10 times as high as that of the naturalone.

For improving the efficiency of absorption of the GLP-1 derivative ofthe present invention, a charge-regulated fat emulsion described inJP-A8-27018 was used to make a preparation. That is, the presentinvention also provides a GLP-1 preparation comprising the fat emulsionregulated to be negatively charged thereon and the GLP-1 derivative ofthe present invention.

The charge-regulated fat emulsion is a fat emulsion regulated to benegatively charged thereon, which is considered to absorb a peptide andprotein thereby improving the stability of the peptide and proteinagainst enzymes, simultaneously enhancing its pharmacological effect andextending duration. The GLP-1 derivative of the present invention, onone hand, has positively charged residues of arginine or lysine addedthereto, and thus easily adheres to the charge-regulated fat emulsion.Accordingly, it is estimated that the mucous membrane absorption of theGLP-1 derivative of the present invention is increased when itspreparation is produced by using the charge-regulated fat emulsion. Whenthe efficiency of absorption of the GLP-1 derivative was actuallyexamined by administering the preparation containing the GLP-1derivative used in combination with the charge-regulated fat emulsion tomice in a glucose tolerant test and then measuring its blood glucoselowering activity in the mice, the GLP-1 derivative of the presentinvention in an amount of 1/30 relative to the naturally occurring GLP-1exhibited an effect similar to the natural one. That is, it isconsidered that the absorption of the GLP-1 derivative of the presentinvention via the nasal mucous membrane, when used in combination withthe charge-regulated fat emulsion, is 30 times as high as that of thenaturally occurring GLP-1.

As described above, the GLP-1 derivative of the present invention is themost suitable peptide for making a preparation having high absorptivityvia mucous membranes, in particular, via the nasal mucous membrane. TheGLP-1 derivative of the present invention, upon substitution at position8 with serine, is made hardly degradable with dipeptidyl peptidase IVoccurring in blood and tissues, thus giving a GLP-1 derivative having alonger half-life in the living body. By conferring trypsin resistance asdescribed above, the GLP-1 derivative can be protected againstdegradation with trypsin or trypsin-like enzymes and so on occurring intissues, thus further improving bioavailability.

By combination with the charge-regulated fat emulsion, the GLP-1derivative of the present invention can improve mucous membraneabsorptivity to exhibit its effect in such a low dose as in aninjection. That is, the present invention is to significantly improvethe clinical applicability of a mucous membrane absorption-type GLP-1preparation, which can be easily administered into patients withoutpain, to improve the quality of life of patients with diabetes andpatients with obesity.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. GLP-1(7-35) is a peptide having the following sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly(SEQ ID NO: 1). Herein, [Ser⁸] means that amino acid at position 2 inthis sequence, that is, Ala at position 8 is replaced by Ser, and [Ser⁸]has the same meaning as 8S. Further, —NH₂ indicates that the sequence inquestion is amidated, and the GLP-1 derivative of the present inventioncan take a form amidated or not amidated at the C-terminus thereof.

The GLP-1 derivative of the present invention can be produced bychemical synthesis or genetic recombination techniques.

The principle of chemical synthesis of polypeptides is known in thetechnical field of the present invention. For this principle, referencecan be made to the following general texts: Dugas H. and Penney C.,“Bioorganic Chemistry” (1981) Springer-Verlag, New York, pp. 54-92;Merrifields J M, Chem. Soc., 85: 2149 (1962); and Stewart and Young,“Solid Phase Peptide Synthesis”, items 24-66, Freeman (San Francisco,1969). For example, 430A peptide synthesizer (PE-Applied BiosystemsInc., 850 Lincoln Center Drive, Foster City Calif. 94404) and asynthetic cycle supplied by PE-Applied Biosystems can be used tosynthesize the peptide of the present invention by the solid phasemethod. Boc amino acids and other reagents can be purchased fromPE-Applied Biosystems and other chemical suppliers.

Hereinafter, the method of producing the peptide of the presentinvention by genetic recombination techniques will be described indetail.

The DNA of GLP-1 is obtained by synthesis or by modifying DNA encoding alarger naturally occurring glucagon. A DNA sequence encoding apreproglucagon is shown by Lund et al. (Proc. Natl. Acad. Sci. USA 79:345-349 (1982)), and this naturally occurring sequence can be changed toproduce the compound of the present invention. The method ofconstructing the synthetic gene is known in the technical field of thisinvention, and reference can be made to, for example, Brown et al.:“Methods in Enzymology”, 68: 109-151, Academic Press, NY. A DNA sequenceencoding the peptide of the present invention is designed on the basisof the amino acid sequence of the peptide, and DNA having the DNAsequence can be produced by a usual DNA synthesizer such as Model 380Aor 380B DNA synthesizer (PE-Applied Biosystems Inc., 850 Lincoln CenterDrive, Foster City Calif. 94404).

The DNA used in production of the GLP-1 derivative of the presentinvention can be devised to increase its expression level and accumulatethe product stably in a host, devised to facilitate purification afterproduction, or devised to produce a fusion protein from which the GLP-1derivative can be easily cut off. For example, such techniques can beexemplified by a technique that involves linking a plurality of genes intandem each encoding the GLP-1 derivative of the present invention, toincrease the expression level, and a technique that involves linking thegene to a gene of protein such as β-galactosidase, β-lactamase, proteinA or TrpE to produce the GLP-1 derivative as a fusion protein. In thesecases, the GLP-1 derivative can be obtained as its sole peptide afterproduction by previously inserting a gene corresponding to an amino acidmethionine into between the respective genes and treating the productwith cyan bromide. In this case, the C-terminus of this product is Hse(homoserine). Some GLP-1 derivatives of the present invention havearginine at the C-terminus only, and can be enzymatically treated witharginyl endopeptidase to give the GLP-1 derivative as its sole peptide.

For efficiently expressing the GLP-1 derivative peptide, its syntheticDNA having a predetermined sequence is inserted into a suitablerecombinant DNA expression vector by suitable restriction endonuclease.Generally, reference can be made to a literature of Maniatis et al.(“Molecular Cloning”, A Laboratory Manual, Vols. 1-3 (1989), ColdSprings Harbor Laboratory Press, NY). To achieve efficient transcriptionof the synthetic gene, the gene is operatively linked to apromoter/operator region. The synthetic gene promoter/operator region isarranged in the same sequence orientation with respect to the ATGinitiation codon of the synthetic gene. Various expression vectorsusable in transformation of procaryotic cells and eucaryotic cells areknown, and reference can be made to “The Promega Biological ResearchProducts and Catalogue” and “The Stratagene Cloning Systems Catalogue”.

After construction of the expression vector for the GLP-1 derivativepeptide, the vector is used to transform suitable host cells. As thehost cells, either eucaryotic or procaryotic cells can be used.Techniques for transforming the cells are known in this field, and canbe found in general literatures such as the above literature of Maniatiset al. Generally, the procaryotic host cells produce a protein in ahigher ratio and can be cultured more easily. A protein expressed in ahigh-level microbial expression system is uniquely aggregated to formparticles or inclusion bodies containing the protein expressed in excessat high level. Such typically aggregated protein is solubilized bytechniques known in this field, denatured and folded again. For suchtechniques, reference can be made to “Protein Folding”, Kreuger et al.(1990), pp. 136-142, ed. Gierasch and King, American Association forAdvancement of Science Publication.

The GLP-1 derivative of the present invention can be combined with apharmaceutically acceptable carrier, diluent, excipient or absorptionpromoter to prepare a pharmaceutical composition. The absorptionpromoter includes, for example, a chelating agent (for example, EDTA,citric acid, salicylate), a surfactant (for example, sodiumdodecylsulfate (SDS)), a non-surfactant (for example, unsaturated cyclicurea) and bile acid salts (for example, sodium deoxycholate, sodiumtaurocholate). Such pharmaceutical compositions can be produced bymethods known in the field of pharmaceutical manufacturing. Thesepharmaceutical preparations are suitable for administration via a mucousmembrane in the nasal cavity or the like, and can be used alone or incombination with other therapeutic agents. The GLP-1 derivative of thepresent invention can also be formed into injections, oral preparationsand the like other than the preparation for mucous membraneadministration.

Using methods known in the technical field of the present invention, thecomposition of the present invention can be formed into a preparation toprovide continuous or sustained release of the active ingredientimmediately after administration into patients. For example, suitablemacromolecules (for example, polyester, polyamino acid, polyvinylpyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethylcellulose and protamine sulfate), or a polymer substance such aspolyester, polyamino acid, hydrogel, poly(lactic acid) or ethyl vinylacetate copolymer can be used to form a complex of the peptide of thepresent invention or to absorb the peptide of the present invention, inorder to produce a preparation showing controlled release. Instead ofmixing the peptide with particles of these polymers, the peptide of thepresent invention can be encapsulated in microcapsules produced bycoacervation techniques or interfacial polymerization, microcapsulesincluding hydroxymethyl cellulose or gelatin, in colloidal drug deliverysystem (for example, liposomes, albumin, microspheres, microemulsion,nano-particles and nano-capsules) or in microemulsion.

In the present invention, a preparation with further improvement inabsorption of the peptide of the present invention via mucous membranescan be produced by absorbing the peptide of the present invention onto acharge-regulated fat emulsion prepared according to JP-A 8-27018. As thecharge regulator, at least one substance selected from various acidicphospholipids and salts thereof, various fatty acids and salts thereof,bile acids and salts thereof is used. The acidic phospholipids and saltsthereof include, but are not limited to, phosphatidyl serine,phosphatidyl glycerol, phosphatidyl inositol, phosphatidic acid andsalts thereof. The fatty acids and salts thereof are not particularlylimited either, but are preferably C6 or more fatty acids and saltsthereof. The bile acids and salts thereof include, but are not limitedto, dehydrocholic acid, deoxycholic acid, taurocholic acid and saltsthereof. By selecting the charge regulator and establishing theconcentration of the charge-regulated fatty emulsion, the pharmaceuticalcomposition of the present invention suitable for administration sitecan be prepared.

The GLP-1 derivative of the present invention is effective againstvarious diseases against which a GLP-1 preparation is effective. Thatis, the GLP-1 derivative of the present invention can be used to treatnon-insulin dependent mellitus diabetes, insulin-dependent mellitusdiabetes, obesity and/or suppression of appetite.

The dose of the GLP-1 derivative of the present invention is determineddesirably by those skilled in the art, depending on the respectivepatients with various diseases. Generally speaking, however, the amountof the GLP-1 derivative to be administered once is considered to be inthe range 1 μg/kg to 1 mg/kg, preferably 10 μg/kg to 100 μg/kg. Justbefore a meal, the GLP-1 derivative is administered once to thrice ormore times every day.

Hereinafter, the present invention is described in more detail byreference to Examples and Test Examples. However, these examples are notintended to limit the scope of the present invention.

PRODUCTION EXAMPLE Synthesis of GLP-1 Derivatives

Synthesis of GLP-1 derivative was conducted by solid phase synthesiswith Model 430A Peptide Synthesizer (PE-Applied Biosystems, Foster City,Calif.), and the products were purified by HPLC, and the synthesizedproducts were confirmed by mass spectrometry. The purity of a majorityof samples was 95% or more, and such samples were used in in vitro andin vivo tests.

Hereinafter, the synthesized compounds are shown. The sequence of GLP-1(7-36) isHis-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2) (that is, GLP-1 (7-36) is the same as GLP-1(7-35)-Arg). For example, GLP-1 (7-36)+Arg-NH₂ is a compound comprisingnaturally occurring G-GLP-1 (7-36) having one amidated Arg residue addedto the C-terminus thereof. Moreover, [Ser⁸]-GLP-1 (7-35) is a compoundwherein Ala at position 2 (corresponding to position 8) was replaced bySer and the last (corresponding to position 36) Arg was deleted.

Comparative Production Example 1 GLP-1 (7-36)—NH₂, which is theNaturally Occurring GLP-1

Comparative Production Example 2: [Ser⁸]-GLP-1 (7-35)-Arg-NH₂ (SEQ IDNO: 3), which is abbreviated to 8S-GLP-1

Production Example 1 GLP-1 (7-36)+Arg-NH₂ (SEQ ID NO: 4), which isAbbreviated to GLP-1+1R Production Example 2 GLP-1 (7-36)+Arg-Arg-NH₂(SEQ ID NO: 5), which is Abbreviated to GLP-1+2R Production Example 3[Ser⁸]-GLP-1 (7-35)-Arg-Arg-Arg-NH₂ (SEQ ID NO: 6), which is Abbreviatedto 8S-GLP-1+2R Production Example 4 [Ser⁸]-GLP-1(7-35)-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 7), which is Abbreviated to8S-GLP-1+3R Production Example 5 [Ser⁸]-GLP-1(7-35)-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 8), which is Abbreviated to8S-GLP-1+4R Production Example 6 [Ser⁸]-GLP-1(7-35)-Arg-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 9), which is Abbreviatedto 8S-GLP-1+5R Production Example 7 [Ser⁸]-GLP-1(7-35)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 10), which isAbbreviated to 8S-GLP-1+6R Production Example 8 [Ser⁸]-GLP-1(7-35)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 11), which isAbbreviated to 8S-GLP-1+8R Production Example 9 [Ser⁸]-GLP-1(7-35)-Lys-Arg-NH₂ (SEQ ID NO: 12), which is Abbreviated to 8S,des36R-GLP-1+1KR Production Example 10 [Ser⁸]-GLP-1(7-35)-Lys-Lys-Arg-NH₂ (SEQ ID NO: 13), which is Abbreviated to 8S,des36R-GLP-1+2KR Production Example 11 [Ser⁸]-GLP-1(7-35)-Lys-Lys-Lys-Arg-NH₂ (SEQ ID NO: 14), which is Abbreviated to 8S,des36R-GLP-1+3KR Production Example 12 [Ser⁸]-GLP-1(7-35)-Lys-Lys-Lys-Lys-Lys-Arg-NH₂ (SEQ ID NO: 15), which is Abbreviatedto BS, des36R-GLP-1+5KR Production Example 13 [Ser⁸]-GLP-1(7-35)-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Arg-NH₂ (SEQ ID NO: 16), which isAbbreviated to 8S, des36R-GLP-1+7KR Production Example 14 [Ser⁸]-GLP-1(7-35)-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Arg-NH₂ (SEQ ID NO: 17),which is Abbreviated to 8S, des36R-GLP-1+10KR Production Example 15[Ser⁸]-GLP-1 (7-35)-Arg-Lys-Lys-NH₂ (SEQ ID NO: 18), which isAbbreviated to 8S-GLP-1+2K Reference Production Example 1 [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1 (7-35)-Arg (SEQ ID NO: 19), which is Abbreviated to8S26Q34N-GLP-1 Reference Production Example 2 [Gln²⁶, Asn³⁴]-GLP-1(7-35)-Arg-NH₂ (SEQ ID NO: 20), which is Abbreviated to 26Q34N-GLP-1

Besides these production examples, GLP-1 derivatives such as ProductionExample 16, [Ser⁸, Gln²⁶ Asn³⁴]-GLP-1 (7-35)-Arg-Arg-Arg-Arg-Arg-NH₂(SEQ ID NO: 21) (abbreviated to 8S26Q34N-GLP-1+4R); Production Example17, [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-35)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH₂(SEQ ID NO: 22) (abbreviated to 8S26Q34N-GLP-1+6R); and ProductionExample 18, [Ser⁸ Gln²⁶ Asn³⁴]-GLP-1 (7-35)-Lys-Lys-Lys-Lys-Lys-Arg-NH₂(SEQ ID NO: 23) (abbreviated to 8S26Q34N, des36R-GLP-1+5KR) areconsidered preferable.

With respect to Production Examples 1 to 15 and Production Example 16 to18, the C-terminus is amidated (—NH₂), but such derivatives can benon-amidated (—OH) derivatives. For example, the non-amidated (—OH)derivative in Production Example 5 can be made the derivative inProduction Example 19, that is, [Ser⁸]-GLP-1 (7-35)-Arg-Arg-Arg-Arg-Arg(SEQ ID NO: 24) (abbreviated to 8S-GLP-1+4R). The C-terminus can be Hse.Such peptide can be exemplified by the derivative in Production Example20, that is, [Ser⁸]-GLP-1 (7-35)-Arg-Arg-Arg-Arg-Hse (SEQ ID NO: 25)(abbreviated to 8S-GLP-1+3RHse).

Test Example 1 Cyclic AMP-Producing Activity of GLP-1 Derivatives

An expression vector of GLP-1 receptor was constructed on the basis of apublished DNA sequence of human GLP-1 receptor (Graziano et al., BiochemBiophys Res Com 196: 141-146 (1993)). Chinese hamster ovary CHO-K1 cellswere transformed with the vector to give recombinant CHO-K1 cellsexpressing human GLP-1 receptor.

The human GLP-1 receptor-expressing cells were put at a density of 1×10⁴cells/ml/well on a 24-well plate. After 3 days, the cells were used inassay and incubated together with each GLP-1 derivative at 37° C. for 30minutes in a buffer (PBS, 5.6 mM glucose, 1 mM isobutyl methyl xanthine,20 μM Ro20-1724, 0.5% BSA, pH 7.4). 10 μl of 5 N hydrochloric acid wasadded to terminate the incubation.

Cyclic AMP products formed in the cells by the reaction of various GLP-1derivatives with the GLP-1 receptor were measured by enzyme immunoassayswith cAMP-Screen system (Applied Biosystems). The cyclic AMP-producingactivities of the various GLP-1 derivatives, in terms of relativeactivity to that (=100%) of naturally occurring GLP-1, are shown inTable 1.

TABLE 1 Cyclic AMP-producing activities of various GLP-1 derivatives, interms of relative activity to that (=100%) of naturally occurring GLP-1,in receptor-expressing cells Concentration of GLP-1 derivative (M) GLP-1derivatives 1 × 10⁻¹¹ 1 × 10⁻¹⁰ × 10⁻⁹ GLP-1 100.0 100.0 100.0 GLP-1 +1R 7.4 100.6 78.1 GLP-1 + 2R 72.3 91.9 112.4 8S-GLP-1 97.9 111.4 83.68S-GLP-1 + 2R 56.4 95.5 91.2 8S-GLP-1 + 3R −17.0 75.6 101.9 8S-GLP-1 +4R 34.0 64.7 105.5 8S-GLP-1 + 5R 54.3 48.6 75.6 8S-GLP-1 + 6R 64.9 81.180.6 8S-GLP-1 + 8R 74.5 87.4 95.9 8S, des36R-GLP-1 + 1KR 100.1 112.980.5 8S, des36R-GLP-1 + 2KR 36.2 88.9 81.6 8S, des36R-GLP-1 + 3KR 45.096.6 86.7 8S, des36R-GLP-1 + 5KR 7.9 55.3 63.4 8S, des36R-GLP-1 + 7KR8.8 55.6 92.6 8S, des36R-GLP-1 + 10KR 7.5 20.8 55.4 8S-GLP-1 + 2K 53.2103.8 88.8

As a result, any GLP-1 derivatives had an in vitro activity of producingcyclic AMP. However, there was a tendency for the activity to bedecreased as the number of arginine or lysine residues added wasincreased. This tendency was recognized particularly with respect tolysine. However, it is highly possible that the added arginine or lysineresidues will be cleaved by peptidase upon absorption via a mucousmembrane, and thus this in vitro activity does not necessarily reflectthe in vivo activity, and this in consistence may bring about results inin vivo tests described below.

Test Example 2 Blood Glucose Lowering Activity and Insulin SecretionStimulating Activity of GLP-1 Derivatives Absorbed Via Mucous Membrane

An increase in absorption of the GLP-1 derivative via a mucous membranewas evaluated in terms of in vivo blood glucose lowering activity andinsulin secretion stimulating activity. That is, the GLP-1 derivativewas administered via the nose into mice, and evaluated in an oralglucose tolerance test (OGTT) where a change in blood glucose level inthe mice after tolerated with glucose was measured.

The GLP-1 derivative was dissolved in distilled water to give 1 mMsolution which was then stocked at −80° C. At the time of the test, thesolution was diluted to a predetermined concentration with physiologicalsaline.

Mice were anesthetized lightly with ether. 20 μl GLP-1 derivativesolution was taken in a micropipette and discharged through the tip ofthe micropipette into the nose of each mouse. The GLP-1 derivativesolution was inhaled by breathing through the nose into the mouse. Fiveminutes after the nasal administration of GLP-1 derivative, 5% glucosesolution was orally administered through a probe in an amount of 10ml/kg.

The blood glucose level was determined by collecting several μl bloodfrom a cut in the tail just before the test and 5, 10 and 20 minutesafter glucose administration, and measuring the blood with a smallblood-glucose-level measuring instrument (Glutest Ace, manufactured bySanwa Kagaku Kenkyusho Co., Ltd.). The area under the bloodconcentration-time curve (AUC 0-20 min) as an increase from the bloodglucose level in each mouse before administration of the GLP-1derivative was calculated.

The blood insulin level was determined by collecting 75 μl blood from anorbital venous plexus with a heparin-treated glass capillary 5 minutesafter administration of glucose, then centrifuging the blood andmeasuring plasma insulin by EIA (Levis Mouse Insulin Kit, manufacturedby Shibayagi Co., Ltd.).

The blood glucose level and blood insulin level in the group given GLP-1derivatives, expressed in terms of average and standard error, are shownin Table 2.

TABLE 2 Blood glucose level and blood insulin level upon administrationof GLP-1 derivative in OGTT test Blood glucose Blood insulin level (areaunder level (5 blood concentra- minutes after Dose tion-time curveglucose (nmol/ (AUC_(0-20 min)) adminis- Administered sample mouse)(mg/dl · min) tration (ng/mL) Physiological saline 0  712 ± 103  632 ±107 Naturally occurring GLP-1 1.07 697 ± 94 662 ± 91 Naturally occurringGLP-1 10.7 399 ± 35  743 ± 147 8S-GLP-1 1.07 624 ± 89 1026 ± 1998S-GLP-1 10.7 291 ± 66 1289 ± 165 8S-GLP-1 + 2R 1.07 535 ± 58 957 ± 868S-GLP-1 + 3R 1.07 509 ± 92 1359 ± 318 8S-GLP-1 + 4R 1.07 388 ± 54 1713± 430 8S-GLP-1 + 5R 1.07 483 ± 26 1504 ± 250 8S-GLP-1 + 6R 1.07 559 ± 271633 ± 449 8S-GLP-1 + 8R 1.07 487 ± 32 1882 ± 402 8S, des36R-GLP-1 + 1KR1.07 611 ± 51 1349 ± 244 8S, des36R-GLP-1 + 2KR 1.07 564 ± 52 1243 ± 3098S, des36R-GLP-1 + 3KR 1.07 557 ± 53 1176 ± 233 8S, des36R-GLP-1 + 5KR1.07 404 ± 71 2229 ± 346 8S, des36R-GLP-1 + 7KR 1.07 457 ± 69 1604 ± 3448S, des36R-GLP-1 + 10KR 1.07  492 ± 106 2379 ± 520 8S-GLP-1 + 2K 1.07598 ± 63  862 ± 150

As a result, it was found that GLP-1 derivatives 8S-GLP-1+4R and 8S,des36R-GLP-1+5KR exhibited the strongest blood glucose depressingaction. The derivatives having arginine residues added to 8S-GLP-1+4R ormore or the derivatives having lysine residues added to 8S,des36R-GLP-1+5KR or more exhibited the strongest insulin secretionstimulating activity. These derivatives in an amount of 1/10 relative tonaturally occurring GLP-1 showed a similar effect to that of thenaturally occurring one, and thus the absorption of these GLP-1derivatives was considered to be 10 times as high as that of thenaturally occurring GLP-1.

Test Example 3 Action of Charge-Regulated Fat Emulsion on Absorption ofGLP-1 Derivatives Via Mucous Membrane

The blood glucose lowering activity and insulin secretion stimulatingactivity of GLP-1 derivatives used in combination with thecharge-regulated fat emulsion were examined in an oral glucose tolerancetest conducted in the same manner as in Text Example 2. Thecharge-regulated fat emulsion used was prepared according to JP-A8-27018; that is, a charge-regulated fat emulsion at a finalconcentration of 8% was obtained by using 2% (w/w) phosphatidyl glycerol(sodium salt), 8% (w/w) neutral oil and 90% (w/w) water.

50 μl of 8% charge-regulated fat emulsion was mixed with 3.56 μl of 1 mM8S-GLP-1+5R solution and 146.4 μl distilled water to give 2%charge-regulated fat emulsion solution containing 8S-GLP-1+5R at a finalconcentration of 0.0178 mM. As controls for comparison, 0.534 mMnaturally occurring GLP-1 solution (whose concentration is 30 times ashigh as that of 8S-GLP-1+5R) not containing 2% charge-regulated fatemulsion, 0.0178 mM 8S-GLP-1+5R solution not containing 2%charge-regulated fat emulsion, and physiological saline were usedrespectively.

Mice were anesthetized lightly with ether. 20 μl GLP-1 derivativesolution was taken in a micropipette and discharged through the tip ofthe micropipette into the nose of each mouse. The GLP-1 derivativesolution was inhaled by breathing through the nose into the mouse. Fiveminutes after the nasal administration of GLP-1 derivative, 5% glucosesolution was orally administered through a probe in an amount of 10ml/kg.

The blood glucose level was determined by collecting several μl bloodfrom a cut in the tail just before the test and 10, 20 and 30 minutesafter glucose administration, and measuring glucose with ablood-glucose-level measuring instrument (Glutest Ace, manufactured bySanwa Kagaku Kenkyusho Co., Ltd.). The area under the bloodconcentration-time curve (AUC 0-30 min) as an increase from the bloodglucose level in each mouse before administration of the GLP-1derivative was calculated.

The blood insulin level was determined by collecting 75 μl blood from anorbital venous plexus with a heparin-treated glass capillary 10 minutesafter administration of glucose, then centrifuging the blood andmeasuring plasma insulin by EIA (Levis Mouse Insulin Kit, manufacturedby Shibayagi Co., Ltd.).

The blood glucose level and blood insulin level in the group given GLP-1derivatives, expressed in terms of average and standard error, are shownin Table 3.

TABLE 3 Blood glucose level and blood insulin level in OGTT test uponadministration of GLP-1 derivatives used in combination with charge-regulated fat emulsion Blood glucose Blood level (area under insulinlevel blood concentra- (10 minutes Dose tion-time curve after glucose(nmol/ (AUC_(0-30 min)) administra- Administered sample mouse) (ng/dl ·min) tion) (ng/mL) Physiological saline 0 2122 ± 198 251 ± 12 Naturallyoccurring 10.7 1533 ± 111 377 ± 41 GLP-1 8S-GLP-1 + 5R 0.357 2025 ± 176506 ± 34 Used in combination with charge-regulated fat emulsion8S-GLP-1 + 5R 0.357 1526 ± 354 560 ± 81

As a result, 8S-GLP-1+5R used in combination with the charge-regulatedfat emulsion, in an amount of 1/30 relative to the naturally occurringGLP-1 without charge-regulated fat emulsion, exhibited a similar bloodglucose lowering activity similar to that of the naturally occurringGLP-1. That is, the effect of 8S-GLP-1+5R was exhibited at a lowerconcentration thereof by using the charge-regulated fat emulsion.

Test Example 4 Evaluation of Activity of GLP-1 Derivative 8S26Q34N-GLP-1

The in vitro activity of GLP-1 derivative 8S26Q34N-GLP-1 in producingcyclic AMP was measured according to the method in Test Example 1. Afteramino acid substitution, the activity was maintained as well (Table 4).

TABLE 4 Cyclic AMP producing activity of 8S26Q34N-GLP-1 Concentration ofAmount of cAMP 8S26Q34N-GLP-1 produced (log M) (pmol/10⁵ cell/30 min)−12 0.6 −11 4.7 −10 24.2 −9 76.1 −8 79.2

When the insulin secretion stimulating activity was examined for 30minutes in the presence of 16.7 mM glucose (high blood glucosecondition) by using mouse Langerhans islet, the GLP-1 derivativeS26Q34N-GLP-1 showed a stronger insulin secretion stimulating activitythan that of the naturally occurring GLP-1 (Table 5).

TABLE 5 Insulin secretion stimulating activity of 8S26Q34N- GLP-1 in thepresence of 16.7 mM glucose by using mouse Langerhans islet Assay Amountof insulin secreted concentration (ng/islet/30 min) (log M) Naturallyoccurring GLP-1 8S26Q34N-GLP-1 0 2.61 −10 4.11 3.64 −9 5.11 6.61 −8 6.719.85

The blood glucose lowering activity in mice was examined byadministering the GLP-1 derivative subcutaneously into mice, and 0.5g/kg glucose administered through a tail vain into mice 5 minutes afteradministration of the GLP-1 derivative. The GLP-1 derivative8S26Q34N-GLP-1 showed concentration-dependent depression in bloodglucose level, and this activity was stronger than that of the naturallyoccurring GLP-1 (Table 6)

TABLE 6 Activity of 8S26Q34N-GLP-1 derivative on blood glucose level inmouse Blood sugar level 20 minutes after tolerance with glucose (mg/dl)Dose (μg/kg) Naturally occurring GLP-1 8S26Q34N-GLP-1 0 123 5 119 110 10103 95 20 75 21

The results of this test indicate that the GLP-1 derivative in ReferenceProduction Example 1 maintains a GLP-1 activity. When the test resultsin Table 1 are also taken into consideration, it can be concluded thateven if several residues of arginine and/or lysine are added to theC-terminus of this GLP-1 derivative, its GLP-1 activity can be stillmaintained.

Test Example 5 Evaluation of Resistance of GLP-1 Derivative 8S-GLP-1 toDipeptidyl Peptidase IV (DPPIV)

500 μM GLP-1 derivative 8S-GLP-1 was mixed with 40 μU/μl dipeptidylpeptidase IV and incubated at 37° C. for 60 minutes. Thereafter, thesample was extracted with a 2-fold excess volume of ethanol, centrifugedand evaporated into dryness by an evaporator. The resulting driedproduct was dissolved in distilled water containing 1% BSA, and itscyclic AMP production activity was measured according to Test Example 1,to calculate the residual activity (%).

As a result, there was no difference in activity of the derivativeregardless of whether it was treated or not treated with dipeptidylpeptidase IV, thus indicating that this GLP-1 derivative is resistant todipeptidyl peptidase IV. Accordingly, the GLP-1 derivative can acquireresistance to dipeptidyl peptidase IV by substituting position 8 withserine (Table 7).

TABLE 7 Residual activity (%) DPP IV Peptide − + 8S-GLP-1 100% 101%Naturally occurring GLP-1 100%  25%

Test Example 6 Evaluation of Resistance of GLP-1 Derivative 26Q34N-GLP-1to Trypsin

The GLP-1 derivative 26Q34N-GLP-1 in Reference Production Example 2 wasdissolved at a concentration of 500 μg/ml in 50 mM ammonium bicarbonateat pH 7.8. Then, 5 μl of 500 μg/ml trypsin (Promega Cat. No. V5113) wasadded to 100 μl of the solution, and the mixture was incubated at 37° C.for 1 hour. The reaction was terminated by adding 1200 μl of 71.5%(final 65%) ethanol, and the reaction solution was centrifuged at 15,000rpm at 4° C. for 5 minutes to recover a supernatant which was thenevaporated into dryness. The dried product was dissolved in distilledwater and measured for cAMP activity by the method in Test Example 1, todetermine the residual activity (%).

As a result, there was no difference in activity of the derivativeregardless of whether it was treated or not treated with trypsin, thusindicating that this GLP-1 derivative is resistant to trypsin (Table 8).

TABLE 8 Residual activity (%) Trypsin Peptide − + 26Q34N-GLP-1 100%94.8%

This result indicates that the GLP-1 derivative is rendered resistant totrypsin by substituting amino acids at positions 26 and 34 in the GLP-1derivative with glutamine and asparagine, respectively. Thus, it can beconcluded that GLP-1 derivatives having several residues of arginineand/or lysine added to the C-terminus thereof also have resistance totrypsin.

INDUSTRIAL APPLICABILITY

Clinical application of GLP-1 by subcutaneous injection is advancing atpresent. This is because GLP-1 is a peptide which cannot be absorbedupon oral administration. The product of the present invention solvesthis problem and enables administration in a form other than injection.Because treatment of diabetes by using GLP-1 will last for a long time,the treatment without repeated injection is greatly advantageous to thepatients.

1. A peptide having a formula of GLP-1 Waa-(xaa)n-Yaa, said peptideconsisting of a GLP-1 peptide, Waa connected to the GLP-1 peptide at theC-terminus of the GLP-1 peptide, (Xaa)n connected to Waa, and Yaaconnected to (Xaa)n (in which Waa is Arg or Lys, Xaa is Lys, n is aninteger of 1 to 9, and Yaa is Arg or Arg-NH₂), wherein the GLP-1 peptideis selected from the group consisting of GLP-1(7-35),[Ser⁸]-GLP-1(7-35), [Gin²⁶, Asn³⁴]-GLP-1(7-35), and [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1(7-35).
 2. The peptide according to claim 1, wherein theGLP-1 peptide is [Ser⁸]-GLP-1(7-35).
 3. The peptide according to claim1, wherein the GLP-1 peptide is [Gln²⁶, Asn³⁴]-GLP-1(7-35).
 4. Thepeptide according to claim 1, wherein the GLP-1 peptide is [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1(7-35).
 5. The peptide according to claim 1, wherein n is aninteger of 3 to
 5. 6. The peptide according to claim 1, which has ahigher efficiency of transmucosal absorption than that of naturallyoccurring GLP-1.
 7. A pharmaceutical composition for transmucosaladministration containing as an active ingredient a peptide having aformula of GLP-1-Waa-(xaa)n-Yaa, said peptide consisting of a GLP-1peptide, Waa connected to the (GLP-1 peptide at the C-terminus of theGLP-1 peptide (Xaa)n connected to Waa, and Yaa connected to (Xaa)n (inwhich Waa is Arg or Lys, Xaa is Lys, n is an integer of 1 to 9, and Yaais Arg or Arg-NH₂), wherein the GLP-1 peptide is selected from the groupconsisting of GLP-1(7-35), [Ser⁸]-GLP-1(7-35). [Gln²⁶,Asn³⁴]-GLP-1(7-35), and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1(7-35).
 8. Thepharmaceutical composition according to claim 7, which is used for nasaladministration.
 9. The pharmaceutical composition according to claim 7,which is used in treatment of non-insulin dependent chronic diabetesmellitus, treatment of insulin dependent chronic diabetes mellitus,treatment of obesity and/or suppression of appetite.
 10. Thepharmaceutical composition according to claim 7, wherein the GLP-1peptide is [Ser⁸]-GLP-1(7-35).
 11. The pharmaceutical compositionaccording to claim 7, wherein the GLP-1 peptide is [Gln²⁶,Asn³⁴]-GLP-1(7-35).
 12. The pharmaceutical composition according toclaim 7, wherein the GLP-1 peptide is [Ser⁸,Gln²⁶, Asn³⁴]-GLP-1(7-35).13. A method for treating non-insulin dependent diabetes muellitus,insulin dependent diabetes mellitus and/or obesity which comprisestransmucosally administering to the patient a pharmaceutical compositioncontaining as an active ingredient a peptide having a formula ofGLP-1-Waa-(xaa)n-Yaa, said peptide consisting of a GLP-1 peptide, Waaconnected to the GLP-1 peptide at the C-terminus of the GLP-1 peptide,(Xaa)n connected to Waa, and Yaa connected to (Xaa)n (in which Waa isArg or Lys, Xaa is Lys, n is an integer of 1 to 9, and Yaa is Arg orArg-NH₂), wherein the GLP-1 peptide is selected from the groupconsisting of GLP-1(7-35), [Ser⁸]-GLP-1(7-35), [Gln²⁶,Asn³⁴]-GLP-1(7-35), and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1(7-35).
 14. The methodaccording to claim 13, wherein the GLP-1 peptide constituting the activeingredient of the pharmaceutical composition is [Ser⁸]-GLP-1(7-35). 15.The method according to claim 13, wherein the GLP-1 peptide constitutingthe active ingredient of the pharmaceutical composition is [Gln²⁶,Asn³⁴]-GLP-1(7-35).
 16. The method according to claim 13, wherein theGLP-1 peptide constituting the active ingredient of the pharmaceuticalcomposition is [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1(7-35).